Signal transduction pathways rely on reversible chemical modifications to relay information within and across cells. Covalent modification of protein substrates by ubiquitin and the ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) contribute to pathways that regulate cellular functions including nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcriptional regulation among many others. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play an integral role in eukaryotic nuclear metabolism and cell cycle control and our studies are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish the basis for Ub/Ubl 1) activation, 2) conjugation by E2 and E3 enzymes, 3) signal transduction through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and factors that constitute ubiquitin and SUMO protein conjugation pathways are conserved so our studies will be broadly relevant and will impact research in other Ub/Ubl-related pathways.